The Rhesus blood group antigens are located on transmembrane erythrocyte proteins encompassing the so-called C, c, E, e and D antigens. Approximately 16% of the Caucasian population is Rhesus D negative (RhD(−)) due to an inherited polymorphism. In addition, multiple genetic and serological variants of RhD exist (divided into category II-VII) of which RhDVI is the most clinically relevant. Since category VI positive red blood cells (RBC) carry fewer of the various epitopes of the D protein than RBC of other categories, RhDVI(+) individuals may form alloantibodies against RBC from other RhD positive (RhD(+)) individuals (Issitt, P. D. and Anstee, D. J., 1998. The Rh Blood Group System, Montgomery Scientific Publications, Durham, N.C., pp. 315-423). {Issitt & Anstee 1998 11809/id}
RhD negativity in itself is not associated with any medical conditions, but has important medical implications when a RhD(−) female carries a RhD(+) or RhDVI(+) fetus or a RhDVI(+) female carries a RhD(+) fetus. Fetomaternal RhD alloimmunization may then occur if fetal erythrocytes enter the maternal circulation, usually perinatally (during delivery), and thereby causes the induction of a maternal anti-RhD antibody response. In subsequent pregnancies RhD-specific IgG-molecules from the mother will cross the placenta into the fetal circulation and mediate lysis of fetal erythrocytes, thereby causing Hemolytic Disease of Newborns (HDN). It has been estimated that on average 20% of RhD(−) women delivering a RhD(+) infant for the second time, and who are not protected appropriately with anti-D prophylaxis, will generate an anti-RhD antibody response. When untreated, approximately 30% of the newborn will have moderate anemia, jaundice, and hepatomegaly, and 20% develop severe anemia and hydrops fetalis, and severely affected newborns are at risk of neonatal death or permanent handicaps.
Polyclonal immunoglobulin preparations against RhD are used worldwide to prevent alloimmunization of pregnant RhD(−) and RhDVI(+) women, thereby preventing hemolytic disease of the newborn. Polyclonal immunoglobulin preparations against RhD (anti-D) are currently obtained by pooling of blood plasma obtained from donors who have become hyperimmune, either through natural RhD alloimmunization or through vaccination of RhD negative volunteer males with RhD positive erythrocytes. The efficacy of anti-RhD immunoglobulin preparations for prophylaxis of HDN is well established and has been in routine use for many years. As a result this severe disease has become a rarity.
Nevertheless the underlying cause of the disease, i.e. alloimmunization of pregnant RhD(−) and RhDVI(+) women, still remains and thus requires a continual supply of anti-D immunoglobulin preparations.
In addition to the prophylaxis of HDN, anti-D immunoglobulin has also proven useful in the treatment of idiopathic thrombocytopenic purpura (ITP) (George, J. N., 2002. Blood Rev. 16, 37-38). ITP is a hematological disorder, where autoantibodies results in an accelerated platelet clearance in the spleen and liver. Symptoms are decreased platelet levels resulting in bruising and bleeding. In severe cases the spleen is removed. This is however, not possible in infants due to severe side effect, thus alternative treatments like anti-D immunoglobulin are needed. Further, anti-D immunoglobulin is used after mistransfusions of RhD(+) blood to RhD(−) recipients in order to prevent sensitization to the Rhesus D antigen.
The current methods for production of anti-D require, as already mentioned, repeated immunization of an increasingly reluctant pool of donors for the production of high titer antiserum. There are also associated risk factors and technical problems, such as the use of Rhesus positive RBC for repeated immunization carrying the risk of transmission of viral diseases like hepatitis B, AIDS and other as yet unknown viruses. Further, there are problems with batch-to-batch variations. Therefore, an alternative method for production of anti-RhD antibodies is required.
Cellular approaches for generating anti-RhD monoclonal antibodies were first developed as an alternative to hyperimmune serum. These techniques encompassed Epstein Barr Virus transformation of lymphocytes creating B lymphoblastoid cell lines (Crawford et al. 1983. Lancet 1, 386-8). However, these cell lines are unstable and require extensive cloning. Production of human antibodies by the hybridoma technique was also restricted by the lack of a suitable human myeloma cell fusion partner (Kozbor, D. and Roder, J. C., 1983. Immunol. Today. 4, 72).
As substitute for these techniques a molecular approach involving repertoire cloning of VH and VL and the construction of phage display libraries was developed (Barbas, C. F. et al. 1991. Proc Natl. Acad. Sci. USA 88, 7978-7982). The phage display technique was also applicable for the isolation of Rhesus D antigen binders. A large number of monoclonal antibodies (mAbs) with Rhesus D antigen binding specificity have been isolated with this technique (WO 97/49809 and Siegel, D. L et al. 2002. Transfus. Clin. Biol. 9, 83-97).
Recent clinical trials with a recombinant anti-RhDVI mAb have shown that it is possible to prevent RhD immunization after a large challenge with RhD(+) RBC (Miescher, S., et al. 2004, Blood 103, 4028-4035). However, the trial also showed that the mAb was less efficient with respect to clearance of the RBC than an anti-D immunoglobulin. The cause of this decreased clearance rate is not known. It is possible that a single antibody is not as efficient as the diversity of antibodies present in the anti-D immunoglobulin product, or that the presence of more than one immunoglobulin isotype i.e. IgG1 and IgG3 {Siegel, Czerwinski, et al. 2002 10320/id} increases RBC clearance.
In addition to the efficiency issue, another issue with respect to HDN prophylaxis is the situation where a RhDVI(+) female carries a RhD(+) fetus. In this situation an anti-RhDVI mAb will not be able to prevent alloimmunization of the female. Thus, in order to protect both RhD(−) and RhDVI(+) females, a product with antibodies against Rhesus D category VI antigen as well as antibodies that do not bind category VI antigen but other common Rhesus D antigens is needed.
Another possible issue with mAbs is that they might be immunogenic. Although the mAbs are human, a first time treatment might result in an antibody response from the female treated with the mAb. Theoretically this may happen because the CDR regions of the mAb, which have never been seen by the immune system of the treated individual before, may be recognized as foreign if presented in a sufficiently large dose. Such a reaction will render the anti-RhD mAb useless in repeated prophylactic treatment.
It is possible that some of these potential problems with mAbs could be overcome by mixing monoclonal antibodies. However, this would mean separate production and purification of an undefined number of antibodies, which will be quite costly. Further, different batch properties of the individual monoclonal antibodies of such a mixture may affect the final product.